Inhibition of proliferation of cells

ABSTRACT

The present invention provides a method of inhibiting the proliferation of cells. The method comprises inhibiting induction or decreasing expression of Egr-1 or decreasing the nuclear accumulation or activity of the Egr-1 gene product. The present invention also provides a method of reducing the incidence of restenosis in a subject. The method comprises administering to the subject an agent which inhibits induction or decreases expression of Egr-1 or decreases the nuclear accumulation or activity of the Egr-1 gene product.

FIELD OF THE INVENTION

The present invention relates to a method of inhibiting the activationof a gene which has in turn been shown to lead to the induction of anumber of other genes that have been strongly implicated in thedevelopment of vascular disease such as atherosclerosis and restenosis.In addition, the present invention relates to oligonucleotides which canbe used in this method. The invention seeks to inhibit the proliferationof cells, migration of cells to sites of injury and remodelling ofvascular wall, associated with the pathogenesis of atherosclerosis orrestenosis, such as smooth muscle cells or endothelial cells.

BACKGROUND OF THE INVENTION

Atherosclerosis is thought to originate from a subtle process ofendothelial injury. Vascular endothelium constitutes a non-thrombogenicsurface of normally quiescent cells that line blood vessels and regulatemolecular and cellular movement across the vessel well. In response todenuding injury, endothelial cells at the wound edge spread and migrateinto the vacant area, undergo proliferation and secrete factors thatstimulate endothelial and smooth muscle cell growth. These responsesprovide an important homeostatic mechanism for maintaining normalvascular function. Growth factors such as platelet-derived growth factor(PDGF, which comprises an A chain and/or a B chain) and basic fibroblastgrowth factor (bFGF) have been implicated to play key roles in theregenerative events following vascular injury. The induction of PDGFgene expression in vascular endothelium may have profound chemotacticand mitogenic effects on the underlying smooth muscle cells andcontribute to the structural remodelling that typically occurs inexperimental arterial repair, restenosis and in the pathogenesis ofatherosclerotic vascular disease (1). Smooth muscle cells are found inboth fatty streaks and fibrous atherosclerotic plaques. Theirproliferation and ability to form enormous amounts of connective tissuematrix and accumulate lipid are key contributing factors in thedevelopment of the atherosclerotic lesion.

Despite a wealth of descriptive studies which correlate the formation ofvascular occlusive lesions with the inappropriate expression of theseand other growth regulatory molecules (2), a direct link between atranscription factor and the induced expression of apathophysiologically relevant gene has not yet been demonstrated in thecontext of arterial injury.

The treatment of occluded coronary arteries currently involves the useof percutaneous transluminal coronary angioplasty (PCTA) or morerecently PCTA in conjunction with the placement of a device known as astent. PCTA is a balloon device that is delivered to the affected sitevia a catheter and following expansion of the balloon results inphysical removal of the blocking plaque or thrombus and enlargement ofthe local vessel area.

The application of the stent, a fenestrated metallic sleeve, addsadditional support to the re-opened vessel and amongst other benefits,prevents the frequency of elastic recoil of the vessel wall. In some ofthe cases of intervention the benefit of the treatment is short livedand the vessel undergoes reclosure or restenosis. Restenosis is amulti-phased clinical event and can involve elastic recoil in the firstinstance followed by extensive vascular remodelling and luminalshrinkage. The final stages of the restenotic process involverecruitment and proliferation of smooth muscle cells to create aneo-intimal mass between the elastic lamina and the endothelium. Theincidence of restenosis has gradually reduced with the advancement ofhealthcare methods but is still a significant problem (Kimura et al.(1996) New England Journal of Medicine 335:561-566, Bittl, (1996) NewEngland Journal of Medicine 334:1290-1302).

There is considerable activity focussed on the development ofpharmaceuticals to be used as adjuncts to the interventional methods inan attempt to reduce the incidence of restenosis. Some of the classes ofdrugs under development include:

(a) Anticoagulants—agents such as hirudin and bivalirudin target theformation of thrombin rich clots.

(b) Antiplatelet drugs—suppression of platelet activation can reduce theformation of platelet aggregates and clotting. One approach involves theuse of a monoclonal antibody dubbed Abciximab that is specific for theplatelet fibrinogen receptor glycoprotein IIb/IIIa.

(c) Antiproliferatives—Trapidil is an antagonist of the PDGF receptor.PDGF is an established stimulator of smooth muscle cell recruitment andproliferation and it is proposed that inhibition of PDGF activity willinhibit this activity.

(d) Antioxidants—compounds such as Probucal are currently underinvestigation as agents to remove oxidative stress from vessel walls andthus limit the smooth muscle cell proliferation associated with suchstress.

(e) Nucleic acid based therapies—antisense and ribozymes directedagainst specific targets e.g. WO 96/25491, WO 96/20279 and WO 96/11266

SUMMARY OF THE INVENTION

It has been found that Egr-1 is rapidly activated following arterialinjury. Induced Egr-1 binds to, and stimulates expression from, thecontrol regions of several genes whose products cause cellproliferation, cell recruitment and vascular wall remodelling ofvascular cells.

Accordingly, in a first aspect, the present invention consists in amethod of inhibiting proliferation of cells comprising inhibitinginduction or decreasing expression of Egr-1 or decreasing the nuclearaccumulation or activity of the Egr-1 gene product.

In a preferred embodiment the cells are vascular cells, particularlysmooth muscle or endothelial cells. The cells may, however, be cellsinvolved in neoplasia.

As will be recognised by those skilled in this field there are a numbermeans by which the method of the present invention may be achieved.These include the following:

(a) Targeting the Egr-1 gene directly using triple helix (triplex)methods in which a ssDNA molecule can bind to the dsDNA and preventtranscription.

(b) Inhibiting transcription of the Egr-1 gene using nucleic acidtranscriptional decoys. Linear sequences can be designed that form apartial intramolecular duplex which encodes a binding site for a definedtranscriptional factor. Evidence suggests that Egr-1 transcription isdependent upon the binding of Sp1. AP1 or serum response factors to thepromoter region. It could be envisaged that inhibition of this bindingof one or more of these transcription factors would inhibittranscription of the Egr-1 gene.

(c) Inhibition of translation of the Egr-1 mRNA using syntheticantisense DNA molecules that do not act as a substrate for RNase H andact by sterically blocking gene expression.

(d) Inhibition of translation of the Egr-1 mRNA by destabilising themRNA using synthetic antisense DNA molecules that act by directing theRNase H-mediated degradation of the Egr-1 mRNA present in theheteroduplex formed between the antisense DNA and mRNA.

(e) Inhibition of translation of the Egr-1 mRNA by destabilisation ofthe Egr-1 mRNA by cleavage of the mRNA by sequence-specific hammerheadribozymes and derivatives of the hammerhead ribozyme such as theMinizymes or Mini-ribozymes or where the ribozyme is derived from:

(i) the hairpin ribozyme,

(ii) the Tetrahymena Group I intron,

(iii) the Hepatitis Delta Viroid ribozyme or

(iv) the Neurospera ribozyme.

The composition of the ribozyme could be:

(i) made entirely of RNA,

(ii) made of RNA and DNA bases,

or

(iii) made of RNA or DNA and modified bases, sugars and backbones

The ribozyme could also be either:

(i) entirely synthetic or

(ii) contained within a transcript from a gene delivered within avirus-derived vector, expression plasmid, a synthetic gene, homologouslyor heterologously integrated into the patients genome or delivered intocells ex vivo, prior to reintroduction of the cells of the patient,using one of the above methods.

(f) Inhibition of translation of the Egr-1 mRNA by cleavage of the mRNAby sequence-specific catalytic molecules composed of DNA. For examplemolecules described previously by Breaker and Joyce (Breaker and Joyce(1995) Chemistry and Biology 2:655-660) could be developed to cleaveEgr-1 mRNA.

(g) Inhibition of Egr-1 activity as a transcription factor usingtranscriptional decoy methods. A method according to that described in(b) above could be used that would interfere with Egr-1 activity andconsequent induction of Egr-1-dependent genes.

(h) Inhibition of the activity of the Egr-1 gene protein by antisenseoligonucleotides that have the potential to hybridise specifically tothe Egr-1 mRNA and contain four consecutive G residues. These G residuesare required for the effect of the oligo in preventing restenosis oratherosclerosis. See WO 96/11266 “Method for inhibiting smooth musclecell proliferation and oligonucleotides for use therein”.

(i) Inhibition of the ability of the Egr-1 gene to bind to its targetDNA by drugs that have preference for GC rich sequences. Such drugsinclude nogalamycin, hedamycin and chromomycin A₃ (Chiang et al J. Biol.Chem. 1996; 271:23999).

In a second aspect the present invention consists in an oligonucleotidefor use in decreasing biosynthesis of Egr-1, the oligonucleotide havingthe sequence ACA CTT TTG TCT GCT (SEQ ID NO:1).

As will be readily recognised by those skilled in the art the process ofrestenosis involves proliferation of smooth muscle cells. Endothelialand smooth muscle cells activated by injury inducibly express geneswhose products are mitogenic and chemotactic to these cells. Accordinglyit is believed that the method of the present invention may haveparticular application in the inhibition or reduction of occurrence ofthis condition.

Accordingly, in a third aspect the present invention consists in amethod of reducing the incidence of restenosis in subject the methodcomprising administering to the subject an agent which inhibitsinduction or decreases expression of Egr-1 or decreases the nuclearaccumulation or activity of the Egr-1 gene product.

As will be understood by those skilled in the art there are a number ofmethods by which the agents which inhibit induction or decreaseexpression of Egr-1 or decrease the nuclear accumulation or activity ofthe Egr-1 gene product may be administered. A useful review of a numberof these delivery routes is provided by Reissen et al (J Am Coll Cardiol1994;223:1234-44) and Wilensky et al (Trends Cardiovasc Med1993:3:163-170), the disclosures of which are incorporated herein byreference.

In particular, delivery of the nucleic acid agents described may beachieved by one or more of the following methods:

(a) Liposomes and liposome-protein conjugates and mixtures.

(b) Using catheters to deliver intra-luminal formulations of the nucleicacid as a solution or in a complex with a liposome.

(c) Catheter delivery to adventitial tissue as a solution or in acomplex with a liposome.

(d) Within a polymer such as Pluronic gels or within ethylene vinylacetate copolymer (EVAc). The polymer will be delivered intra-luminally.

(e) Within a vital-liposome complex, such as Sendal virus.

(f) The nucleic acid may be delivered by a double angioplasty balloondevice fixed to catheter.

(g) The nucleic acid could be delivered on a specially prepared stent ofthe Schatz-Palmaz or derivative type. The stent could be coated with apolymer or agent impregnated with nucleic acid that allows controlledrelease of the molecules at the vessel wall.

As used herein the term DNA refers to primarily to deoxyribonucleotidesit will, however, be readily apparent to those skilled in the art thatderivatives of DNA may be used. It is intended that such derivatives areincluded in the scope of the present invention. The envisagedmodifications are well known to those skilled in the art and include:

(a) phosphodiester backbone modification by replacement of anon-bridging oxygen atom with sulphur or a methyl group such as inphosphorothioates or methylphosphonates or replacement of phosphodiesterbackbone with a peptide linked backbone such as in PNAs.

(b) replacement of the 2′ hydrogen within the deoxyribose group with aamine, methyl or other alkanes or alkenes or other functional group.

(c) modification of the termini of the oligonucleotide by the additionof an inverted base at the 3′ end via 3′—3′ linkages.

(d) modification of 5′ and 3′ by conjugation of other functional groupsselected from lipids and steroids such as cholesterol.

(e) phosphodiester backbone modifications in which the phospho-sugarbackbone is replaced by a morphilino phophorodiamidate backbone.

It will also be understood that similar modifications may be applied tothe RNA oligonucleotides except in (b) the 2′ group that would bereplaced would by hydroxyl.

DETAILED DESCRIPTION OF THE INVENTION

In order that the nature of the present invention may be more clearlyunderstood the preferred forms of the present invention will now bedescribed in greater detail.

FIGURE LEGENDS

FIG. 1 shows uptake of radiolabeled antisense E11 by smooth muscle cells( passive; ▪ “Lipofectamine”).

FIG. 2 shows effect of oligonucleotides (1 μM) on smooth muscle cellproliferation.

In a survey of immediate-early genes that could be induced by acutevascular injury in the rat aorta, the expression of theearly-growth-response gene product Egr-1 (krox-24; NGF-IA, zif268,T1S8), a serum-inducible zinc-finger nuclear phosphoprotein and memberof a family of related transcription factors (3) was examined. In thisexamination aortic endothelium of male Sprague-Dawley rats (400 g) waspartially denuded using an uninflated 2F balloon catheter.Deendothelialized regions were identified by intravenous injection ofEvans blue (0.3 ml of 5% solution in PBS) 10 min prior to sacrifice.Animals were perfusion-fixed with phosphate-buffered 4%paraformaldehyde. Vessel segments were treated with 1 μg/ml proteinaseat 37° C., prehybridized for 2 h at 55° C. in 0.3M NaCl, 20 mM Tris,pH7.5, 5 mM EDTA, 1×Denhardt's, 10% DTT and 50% formamide, and incubatedwith the appropriate ³⁵ S-UTP-labeled riboprobe for 16 h. After washing,the slides were coated with autoradiographic emulsion and exposed for 3wk. The images were photographed and digitized. The hybridization signalof the radiolabeled probe appears as white grains. All specimensobserved under dark field illumination after nuclear counterstain withhematoxylin. Immunostaining for factor-VIII-related antigen confirmedthat injury was limited to endothelium.

In situ hybridization techniques which visualize the endothelium of thevessel wall en face revealed that Egr-1 expression was dramaticallyinduced exclusively at the endothelial wound edge within 30 min ofpartial denudation. Egr-1 expression was undetectable in endotheliumfrom unmanipulated arteries. Induced Egr-1 mRNA was apparent after 2 h,and the time-dependent decrease in the specific hybridization signaldemonstrates the transient induction of endothelial Egr-1 expression byinjury. In contrast, the sense Egr-1 riboprobe failed to hybridize withmRNA from normal or injured tissue. PDGF-B-chain transcript levels werealso low in unmanipulated vessels, consistent with previous findingsusing other techniques (4). Partial denudation did not induce PDGF-bgene expression at the endothelial wound edge until 4 h after injury andcontinued for several weeks during endothelial regeneration (5). Thecolocalization of the spatial patterns of Egr-1 and PDGF-B geneexpression, and the temporal association between these two genes ininjured arterial endothelium, led to a determination of whether Egr-1could inducibly regulate the expression of PDGF-B.

In response to mechanical injury in vitro, confluent endothelial cellsinitiate movement into the open “wounded” area by actively responding tolocally-derived signals or autocoids from injured cells. An in vitromodel of vascular injury (6) was used to address the possible linkbetween Egr-1 and injury-induced PDGF-B gene expression. Nuclear run-offanalysis revealed that Egr-1 gene transcription was induced in culturedbovine aortic endothelial cells (BAEC) within 1 h of injury. 5′ deletionanalysis of the PDGF-B promoter in endothelial cells previously defineda region necessary for core promoter activity (d77) which contained abinding site for the ubiquitous transcription factor, Sp1 (7). Recent invivo footprint analysis of the promoter demonstrates that the Sp1element is indeed occupied in intact cells (8). In vitro DNase Ifootprinting revealed that recombinant Egr-1 protected a regionoverlapping this site from partial DNase I digestion. When nuclearextracts from endothelial cells 1 h after injury were incubated with a³²P-labelled oligonucleotide spanning this region (³²P-Oligo B,5′-GCTGTCTCCACCCACCTCTCGCACTCT-3′ SEQ ID NO:2), a distinct nucleoproteincomplex formed. The injury-induced complex was eliminated by antibodiesto Egr-1. Nuclear Sp1 also bound to the PDGF-B promoter fragment;however, its levels are unaltered by injury. Thus, injury-inducedendothelial Egr-1 expression precedes the induction of PDGF-B, and Egr-1binds to a distinct region in the PDGF-B promoter also bound by Sp1.

The functional importance of this interaction for PDGF-Bpromoter-dependent gene expression was next determined. Northern blotand transient transfection analysis using PDGF-B promoter-reporterconstructs previously revealed that this gene is basally expressed invascular endothelial cells (7). Chloramphenicol acetyltransferase (CAT)expression driven by the PDGF-B promoter (d77-CAT) was induced by injurywithin 36 h. Reporter activity also increased in cells exposed tophorbol 12-myristate 13-acetate (PMA) or by cytomegalovirus-mediatedoverexpression of Egr-1. When a mutation that abolished the ability ofEgr-1 to bind to the PDGF-B promoter was introduced into the d77-CATconstruct, basal expression driven by the promoter was attenuated, andexpression inducible by injury was abolished. The mutant construct alsofailed to mediate increased reporter activity when Egr-1 wasoverexpressed, or when the cells were exposed to PMA. The Egr-1 bindingsite in the proximal PDGF-B promoter is thus required for induciblepromoter-dependent expression in vascular endothelial cells.

The interaction of Egr-1 and Sp1 with overlapping binding elements inthe proximal PDGF-B promoter suggests that Sp1, resident on the promoterin unstimulated cells, may be displaced by increasing levels of Egr-1.Running gel shifts (9) indicate that recombinant Egr-1 bound to thePDGF-B promoter in a stable and reversible manner. The relativeefficiency with which Egr-1 was displaced from ³²P-Oligo B by itsunlabelled counterpart indicates that Egr-1 interacts with the PDGF-Bpromoter with a faster off-rate than its comparable site in the proximalPDGF-A promoter (9). Sp1 was displaced from the promoter by Egr-1 in adose-dependent manner. Decreasing levels of Egr-1 in the presence of afixed concentration of SP1 allowed reoccupation of the promoter by Sp1.The absence of a higher order complex when both factors are presentindicates that Egr-1 and Sp1 do not bind the promoter simultaneously.These findings with recombinant proteins indicate that an interplayinvolving Egr-1 and Sp1 can occur on the PDGF-B promoter.

The localized induction of Egr-1 at the endothelial wound edge precludeda direct determination of whether a displacement mechanism was involvedin the induction of PDGF-B gene expression by injury. PMA is a modelagonist of Egr-1 expression in vascular endothelial cells (9). Thedramatic induction of Egr-1 mRNA and protein that precedes the increasein PDGF-B levels in endothelial cells exposed to PMA is like thetemporal pattern with which these genes are expressed at the endothelialwound edge following arterial balloon injury. Transcript and proteinlevels of Sp1 are also not affected by PMA. Nuclear proteins fromPMA-treated endothelial cells bound to the PDGF-B promoter with apattern similar to that observed using injury-induced extracts.Immunobinding studies determined that nucleoprotein complexes containedeither Sp1 or Egr-1. The profound induction in Egr-1 levels by PMAdemonstrates the ability of this transcription factor to displace Sp1from the PDGF-B promoter in the context of nuclear extracts.Accordingly, the PMA-inducible endothelial expression of the PDGF-Bgene, like PDGF-A (9), involves an interplay between Egr-1 and Sp1 atoverlapping binding sites in the proximal promoter. This contrasts witha previous report suggesting that Egr-1 may serve as a negativeregulator of gene transcription by blocking the binding of Sp1 to itsown recognition sequence (10). These findings suggest that the localizedinduction of PDGF-B expression at the endothelial wound edge may alsoinvolve displacement of promoter-bound Sp1 by elevated levels of nuclearEgr-1. Egr-1 may be involved in interactions with other transcriptionalactivators and the basal complex to mediate increased gene expression inresponse to injury.

Egr-1 also appears to play a key role in injury-inducible PDGFexpression in smooth muscle cells. In the rat arterial injury modelin-situ hybridization with en face preparations indicate that Egr-1expression is induced in smooth muscle cells concurrent with theexpression of PDGF-A at the same location.

Antisense Approach

The antisense approach is based on the ability of an oligonucleotide(synthetic DNA) to recognize its complementary sequence within the cell,in the form of messenger RNA; the bound complex is then able tosterically interfere with ribosome binding and translation into protein(11). Alternatively, the bound complex triggers cleavage of the mRNA bythe nuclease RNase H, which is widely present in mammalian cells andspecifically recognizes DNA-RNA duplexes (12). Thus, the overall effectof a given antisense oligonucleotide may be to reduce specific mRNA andprotein levels if mediated by RNase H, or a reduction in specificprotein levels in the case of steric interference (13).

Advantages offered by the use of antisense oligonucleotides overconventional inhibitors are specificity and synthesis. This is based onthe uniqueness of the target mRNA and the general availability ofoligonucleotide synthesizers. A drawback in this approach is thepropensity of these oligonucleotides to be degraded or inactivated bynucleolytic phosphodiesterases. However, chemical modification of thephosphodiester linkages between individual nucleotides has been found toincrease nuclease resistance by up to ten-fold and increase potency as aconsequence (14,15).

As explained above Egr-1 is an immediate-early gene (16,17) expressed atlow or undetectable levels in arterial endothelial cells (18) or smoothmuscle cells. It is dramatically induced by a number of(patho)physiologically-relevant agonists and conditions such as fluidshear stress, mechanical injury (18), heparin-binding growth factor-1,as well as the protein kinase C-inducer, phorbol 12-myristate 13-acetate(9). Egr-1 mRNA is transcribed and processed in the nucleus; it thenenters the cytoplasm where it is translated to protein. Since Egr-1protein contains a nuclear targeting sequence, it reenters the nucleusand interacts with its nucleotide recognition sequence in the promotersof responsive genes. Two genes which are induced by Egr-1 are thoseencoding the platelet-derived growth factor A-chain (9) and B-chain(18). This growth factor is a potent mitogen and chemoattractant forsmooth muscle cells (19) and produced by cells involved in theatherosclerotic or restenotic lesion. Accordingly, the PDGFligand/receptor signalling system has been implicated in thepathogenesis of atherosclerosis.

Elevated levels of PDGF-A transcripts have been observed in humancarotid plaques (20). The coexpression of PDGF-A with smooth muscleα-actin implicated SMCs in the plaque as a source of PDGF-A (20). Murryand colleagues (21) also found PDGF-A mRNA in human atheroscleroticplaques using competitive RT-PCR. Rekhter and Gordon (22) used a tripleimmunolabeling approach to localize PDGF-A protein to smooth muscle-likecells and some endothelial cells within human carotid plaques. Libby andcolleagues showed that SMCs cultured from human atherosclerotic plaquescould express PDGF-A transcripts and secrete PDGF-like binding andmitogenic activity (23). Barrett and Benditt used Northern blot and dotblot analysis to show that levels of PDGF-B-chain mRNA wereapproximately five-fold greater in carotid plaques than normal aorta andcarotid arteries (24). In situ hybridization later corroborated thesefindings demonstrating that the PDGF-B-chain was associated withendothelium at the luminal surface of the plaque and smooth muscle-likecells within the plaque (25). Ross and coworkers used a doubleimmunostaining technique with carotid endarterectomy specimens to detectPDGF-B protein in macrophages (26).

A number of important considerations were undertaken in the design ofhybridization-specific antisense oligonucleotides to Egr-1. First, eacholigonucleotide was synthesized with a phosphorothioate backbone forincreased stability and potency. Second, oligonucleotides weresize-matched to 15 bases; longer sequences were avoided to reduce thepossibility of non-specific effects seen with longer oligonucleotides(Stein, C. A. (1996) Trends in Biotechnology 14:147-149). Third, asequence of four consecutive guanosines was avoided in light of recentreports indicating that oligonucleotides bearing this sequence, such asthose that have targeted c-myb and c-myc, inhibit proliferation by anonantisense mechanism (27,28). Fourth, the efficacy of multipleoligonucleotides directed toward various regions of Egr-1 mRNA wasassessed. Finally, a size-matched, fully phosphorothioatedoligonucleotide with no sequence complementarity to any portion of Egr-1mRNA was used as a negative control.

Design of Egr-1 antisense oligonucleotides

A panel of antisense oligonucleotides complementary to rat Egr-1 mRNAwere designed to identify regions within the mRNA that were suitabletarget sites. Putative target sites were chosen on the basis of beingencoded within regions of the mRNA that had low secondary structure andtheoretically had a greater potential for inter molecular hybridisation.Such single stranded regions were identified firstly by using the Zukeralgorithm for determination of free energy of RNA molecules (Zuker, M(1989) Science 244:48-52). Once a low energy secondary structure wasdetermined, regions of low frequency intramolecular base-pairing wereidentified by visual examination. The oligo nucleotides used are set outin Table 1

TABLE 1 Nucleotide Sequence of Oligonucleotides (5′->3′) E1 CGC CAT TACCTA GTG (SEQ ID NO:3) A/S2 CTT GGC CGC TGC CAT (SEQ ID NO:4) E6 CCA GGCTGG CGG TAG (SEQ ID NO:5) E7 GAG AAC TGA TGT TGG (SEQ ID NO:6) E9 TGTGGT CAG GTG CTC (SEQ ID NO:7) E11 ACA CTT TTG TCT GCT (SEQ ID NO:1)

Uptake and stability of an Egr-1 antisense oligonucleotide by smoothmuscle cells

One of these oligonucleotides, E11, was radiolabeled with ³²P, andassessed for its ability to associate with cultured vascular smoothmuscle cells. After various times, the cultures were washed withphosphate-buffered saline and removed from the vessel by scraping. Aftercentrifugation, the cells were transferred to Eppendorf tubes,solubilized and either counted in a scintillation counter orelectrophoresed on a denaturing polyacrylamide gel.

Radiolabeled E11 associated with the cells in a time-dependent manner;maximal uptake was observed after 6 h (FIG. 1, passive). Theoligonucleotide was still associated with the cells after 9 h and 24 h(FIG. 1, passive). Electrophoretic analysis indicated that theoligonucleotide did not undergo significant degradation during thecourse of experiment.

Egr-1 antisense oligonucleotides inhibit smooth muscle proliferation

The panel of oligonucleotides were assessed for their ability to inhibitsmooth muscle cell proliferation in an assay of ³H-thymidineincorporation into DNA. Oligonucleotides were added to the culturesupernate 6 h after the medium was changed to serum-free at a finalconcentration of 1 μM and incubated for a further 18 h. The cells werewashed and exposed again to 1 μM oligonucleotide in medium containing aconcentration of serum that would stimulate ³H-thymidine incorporationinto DNA submaximally. After a further 24 h incubation, the cells werepulsed for 6 h with ³H-thymidine prior to the determination ofTCA-precipitable ³H-thymidine incorporation into DNA.

The control oligonucleotide E1, an oligonucleotide of random sequencebearing no complementarity to Egr-1 mRNA, did not alter the rate ofserum-inducible ³H-thymidine incorporation into DNA in smooth musclecells (n=10) (FIG. 2). In contrast, two Egr-1 antisense oligonucleotideswere able to inhibit DNA synthesis. A/S2 and E11, directed againstdifferent portions of Egr-1 mRNA, inhibited by 63% (n=10), and 50%(n=12), respectively (FIG. 2). Trypan blue exclusion studies andmorphologic observations revealed that inhibition was unlikely to be dueto non-specific cytotoxic mechanisms. In contrast, Egr-1 antisenseoligonucleotides E6, E7 or E9 failed to inhibit smooth muscle cellproliferation (FIG. 2). That not every Egr-1 antisense oligonucleotidecould inhibit is consistent with the notion that naturally occurringmRNA has higher order structure and certain sequences may not be asaccessible to certain oligonucleotides as others.

Egr-1 antisense oligonucleotides inhibit Egr-1 protein synthesis, butnot Sp1

Western blot analysis was used to assess the effect of Egr-1 antisenseoligonucleotides on levels of serum-inducible Egr-1. Oligonucleotides ata final concentration of 1 μM, were added to the culture supernates 6 hafter changing the medium to serum-free to render the cells quiescent.After 16 h, the cells were washed with phosphate-buffered saline andincubated with 1 μM oligonucleotide for a further 2 h. The cells werethen exposed to a concentration of serum able to stimulate ³H-thymidineincorporation into DNA submaximally. After 2 h, the cell lysate waselectrophoresed on denaturing polyacrylamide gels prior to transfer andthen assessed for the presence of Egr-1 using specific antibodies.

Serum induced the synthesis of Egr-1 protein within 2 h. Incubation ofthe cells with E1 did not affect the ability of serum to induce Egr-1.In contrast, E11 and A/S2 profoundly inhibited the induction of Egr-1protein. Cellular levels of the related zinc-finger transcriptionfactor, Sp1, were unaffected by either E11 or A/S2 demonstrating thetarget specificity of these oligonucleotides.

Taken together, these findings demonstrate that antisenseoligonucleotides directed to selected regions of Egr-1 mRNA reduce theaccumulation of the Egr-1 protein. Inhibition of Egr-1 is not due tonon-specific or cytotoxic mechanisms. Cells treated with theseoligonucleotides do not undergo morphologic changes or take up Trypanblue. Moreover, whereas Egr-1 protein levels are profoundly attenuatedby these oligonucleotide, levels of the related zinc-fingertranscription factor, Sp1, are unaffected. These oligonucleotides canselectively inhibit smooth muscle cell proliferation.

Discussion

Since Egr-1 is an inducible transcription factor with binding sites inmultiple genes (9,18), these findings strongly suggest that theantiproliferative effects of these oligonucleotides may be due toselective inhibition of the expression of Egr-1-dependent genes requiredfor proliferation, such as PDGF (9,18), bFGF (29) or cell-cycleregulatory genes (30). Egr-1 is not basally expressed in smooth musclecells or endothelial cells of the vessel wall, unless activated bymechanical injury (18). Therefore, Egr-1 is an appropriate target forantiproliferative therapy.

The various oligonucleotides used in these studies were delivered to thecells without a carrier. Uptake is an energy-dependent process and ismaximal at 37° C. (31). Phosphorothioate oligonucleotides have beenfound to associate with an 80 kD protein on the cell surface, consistentwith receptor-mediated endocytosis (32,33). Passive delivery, however,is largely an inefficient process (34). The biologic effects of theseoligonucleotides can be augmented by methodologies which facilitate moreefficient delivery into cells, such as liposomes (35). Indeed, suchapproaches may be useful in increasing the biological potency of E11 andA/S2. The results presented herein indicate that “Lipofectamine”, forexample, can increase both the rate and total uptake of radiolabeled E11(FIG. 2) without affecting the integrity of the oligonucleotide to anysignificant extent. Since post-angioplasty restenosis is usually focal,local delivery of these oligonucleotides may be useful in the treatmentof this disease, which in the United States occurred is approximately30-50% of the over 300,000 procedures performed in 1991 alone (36).

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

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7 1 15 DNA Artificial Sequence Oligonucleotide 1 acacttttgt ctgct 15 227 DNA Artificial Sequence Oligonucleotide 2 gctgtctcca cccacctctcgcactct 27 3 15 DNA Artificial Sequence Oligonucleotide 3 cgccattacctagtg 15 4 15 DNA Artificial Sequence Oligonucleotide 4 cttggccgct gccat15 5 15 DNA Artificial Sequence Oligonucleotide 5 ccaggctggc ggtag 15 615 DNA Artificial Sequence Oligonucleotide 6 gagaactgat gttgg 15 7 15DNA Artificial Sequence Oligonucleotide 7 tgtggtcagg tgctc 15

What is claimed is:
 1. A method of inhibiting proliferation of cellsselected from the group consisting of vascular cells, smooth musclecells, endothelial cells and neoplasia cells, which method comprisesadministering locally to the cells an Egr-1 antisense oligonucleotide—inan amount sufficient to inhibit the proliferation of cells.
 2. A methodaccording to claim 1 in which the cells are smooth muscle cells.
 3. Amethod according to claim 1 in which the oligonucleotide decreasesexpression of Egr-1.
 4. A method according to claim 1 in which theantisense oligonucleotide is selected from the group consisting ofantisense oligonucleotides having the sequence ACA CTT TTG TCT GCT (SEQID NO:1) and CTT GGC CGC TGC CAT SEQ ID NO:4).
 5. An oligonucleotide fordecreasing expression of Egr-1, the oligonucleotide consisting of thesequence ACA CTT TTG TCT GCT (SEQ ID NO:1).
 6. A method of reducing theincidence of restenosis in a subject comprising administering locally tothe subject an Egr-1 antisense oligonucleotide.
 7. A method according toclaim 6 in which the oligonucleotide decreases expression of Egr-1.
 8. Amethod according to claim 6 in which the antisense oligonucleotide hasthe sequence ACA CTT TTG TCT GCT (SEQ ID NO:1) or CTT GGC CGC TGC CAT(SEQ ID NO:4).